In this study, the spatial distribution of the pyridinium/reducible collagen cross-link ratio was determined in thin tissue sections from iliac crest biopsy specimens of patients that had been diagnosed as either high- or low-turnover osteoporotics, as well as premenopausal women with normal BMD sustaining multiple spontaneous fractures. The data were compared with recently reported values encountered in normal bone at equivalent anatomical locations (bone forming trabecular surfaces). The results of this study show for the first time in human subjects that the pyridinium/reducible collagen cross-link ratio differs between normal subjects and patients with fragile bone, even when BMD values are normal.
Fractures, the clinical consequences of osteoporosis, result when bone mass is reduced below a level sufficient to maintain structural integrity. Tissue heterogeneity is a major complicating factor in studies of osteoporosis, many of which use bulk-type analyses of the bony tissues. One of the advantages of FTIR spectroscopic analysis is that it allows for the rapid analysis of thin tissue sections with a spatial resolution of ~6.3 μm, making it possible to examine discrete and anatomically equivalent points.
The organic matrix in osteoporosis has received considerably less attention than the bone mineral. However, accumulated evidence suggests that the matrix content in osteoporotic bone is decreased relative to age- and sex-matched controls and that biochemical alterations are apparent in the collagen molecules.(6
) The intermolecular cross-linking of bone collagen is a chemical feature that is intimately related to the way matrix collagen molecules are arranged in fibrils(15
) and provides fibrillar matrices with important mechanical properties such as tensile strength and viscoelasticity.(24
) The cross-link pattern is determined and influenced by many factors, including the level of lysine hydroxylation,(26
) collagen turnover,(27
) molecular packing structure,(28
) and mineralization,(29
) and has been shown to be specific to the type of tissue(15
) rather than the type of collagen.
In this study, FTIRI was used to examine the cross-linking patterns in bone matrix at bone-forming trabecular surfaces, showing that distinctive differences in the collagen cross-link ratio (pyridinium/reducible) are in fact present. At formative trabecular surfaces of normal bone, the most superficial, that is, the “youngest,” tissue (at the first 6.3 × 6.3-μm measurement point) seemed to lack the mature Pyr cross-links.(18
) This observation is consistent with what is known about the maturation of collagen cross-links based on biochemical analyses.(29
) As the tissue matures (as seen in the next and subsequent 6.3 × 6.3-μm measurement points), complex variations in the cross-link ratios are apparent.
In both the HTOP and LTOP samples, the values of the collagen cross-link ratios were higher than those obtained in the normal samples. This suggests that, in osteoporosis, processes affecting the collagen cross-link ratio in the osteoid matrix are altered, implying that, in addition to the well-established imbalance between formation and resorption seen in osteoporosis, the bone matrix that is produced in osteoporosis is also different from the bone matrix that is made in normal bone. The case of HTOP is similar to that of LTOP, if more dramatic and somewhat enigmatic, because the cross-link ratios become higher more rapidly than in either normal or LTOP samples. However, at ~30 μm from the surface, they become similar to those of normal samples and then again become higher than either the normal or LTOP samples. This variation, which indicates a deviation from the expected sequence of matrix “maturation,” is not readily explained but may become better defined with future knowledge of the mechanism of collagen maturation in these cases, as well as the effect of collagen polymorphisms.(31
Just as intriguing is the data obtained in the SF group of patients. These young women, all under age 40, had normal BMD, serum chemistry, and lacked underlying conditions that might contribute to fractures, yet they spontaneously fractured. The type of analysis presented in this manuscript suggests an identical collagen cross-link profile with the one encountered in low-turnover osteoporotic patients. Whether this is the cause of the skeletal fragility or the result of improper bone repair cannot be determined from this data but importance of bone material properties, a component of bone quality, when considering bone fragility is emphasized.
In summary, FTIRI analysis of thin sections from human iliac crest biopsy specimens revealed differences in the spatial distribution of the pyridinium/reducible collagen cross-link ratio between normal and osteoporotic patients at forming trabecular surfaces. This may be because of the possibility that the matrix produced in osteoporosis matures at a faster rate than in normal bone matrix or the bone matrix of osteoporosis undergoes post-translational modification for a longer period of time than the bone matrix of normal bone, perhaps because of a delay or alteration in mineralization. The fact that similar trends were observed in spontaneously fracturing women with normal BMD accentuate the role of the matrix in determining bone strength and therefore fracture resistance.
As more information concerning details of the matrix is revealed by the use of techniques such as FTIR imaging, the contributing factors to bone fragility can be better understood, and therapeutic protocols can be developed that address issues concerning the quality of the matrix produced, not just its quantity.